1. Methionine Sulfoxide Reductase (Msr)
Oxygen is essential to all aerobic organisms but can also have many harmful effects. Oxidation of Methione residues in a protein or a peptide has been implicated in several serious conditions in humans, including adult respiratory distress syndrome, rheumatoid arthritis, smokers' emphysema, and Alzheimer's disease. There is now growing evidence that enzymatic repair of oxidized Methione residues may play a key protective role in organisms ranging from bacteria to humans (El Hassouni Proc Natl Acad Sci USA. 1999; 96:887-892). In addition to being the most common form of oxidative damage to proteins, the oxidation of Methione to Methionine sulfoxide (MetSO) is unique in being readily reversible by the enzyme peptide-Met sulfoxide reductase (MSR; EC 1.8.4.11), suggesting that MSR may be able to repair oxidatively damaged proteins (Brot et al. Anal Biochem. 1982 b; 122:291-294) in vivo.
The methionine sulfoxide reductase (Msr) family is composed of two monomeric enzymes named MsrA and MsrB, which reduce oxidised methionine residues in a peptide (peptide-L-methionine (S)—S-oxide). MsrA and Msr B display specific stereo-selectivities towards the sulfoxide. Both isoforms contribute and are necessary to protect the cell against the stress caused by the oxidation of Met residues at the sulfur atom which typically present a racemic mixture of the two stereoisomers.
Additionally, MsrA and MsrB types share the same chemical reaction mechanism which includes three steps with (1) formation of a sulfenic acid intermediate with a concomitant release of 1 mol of methionine per mol of enzyme; (2) formation of an intramonomeric disulfide Msr bond followed by; (3) reduction of the oxidized Msr by thioredoxin (Trx). The active sites of both Msrs are adapted for binding protein-bound methionine sulfoxide (MetSO) more efficiently than free MetSO (Boschi-Muller, et al. Biochim. Biophys. Acta (2005); 1703: 231-238; Boschi-Muller et al. 2008, Arch Biochem Biophys. 15; 474(2):266-73). In a number of bacteria, the MSRA and MSRB domains are fused (Kryukov et al. 2002 PNAS 99:4245-4250).
In plant cells, several MSR protein isoforms both of the A and B type are present. The isoforms may be localized to different subcellular compartment such as cytosol, chloroplast or secretory pathway. Phylogenetic analysis revealed that the A and B type have evolved such that two MSR-A and MSR-B subgroups can be distinguished. They differ essentially in the number and in the position
of the cysteines involved in catalysis and enzyme regeneration, but also in the subcellular localization or in the intron/exon distribution of the gene encoding the isoform. MSR-A and MSR-B isoforms contribute to a total MSR enzymatic activity in plant cells as measured by Sanchez at al. 1983 Plant Physiol 73:619-623; Bechtold et al. 2004, Plant Cell 16:908-919. Functionally, the plant MSRs, A and B type included, appear to constitute key components in preventing damage in proteins under severe environmental constraints known to generate-ROS in plastids (Romero et al. 2004 Plant Physiol 136:3784-3794), or under pathogen infection (Sanchez et al. 1983), but also under more subtle treatments such as long night periods (Bechtold et al. 2004).2. Enolase (2-phospho-D-glycerate hydrolase)
Enolase (2-phospho-D-glycerate hydrolase) is an essential glycolytic enzyme that catalyses the interconversion of 2-phosphoglycerate and phosphoenol-pyruvate. Genes encoding Enolase proteins are conserved from prokaryotes to eukaryotes. In vertebrates, isoenzymes alpha, beta and gamma are present: alpha is present in most tissues; beta is localised in muscle tissue; and gamma is found only in nervous tissue. The functional enzyme exists as a dimer of any 2 isoforms: in immature organs and in adult liver, it is usually an alpha homodimer; in adult skeletal muscle, a beta homodimer; and in adult neurons, a gamma homodimer; in developing muscle, it is usually an alpha/beta heterodimer; and in the developing nervous system, an alpha/gamma heterodimer. The tissue specific forms display minor kinetic differences. In plants levels of Enolase transcripts and activity reportedly increased in response to abiotic stresses such as salt, low and high temperature, and anaerobic stresses (Forsthoefel, et al. 1995; Plant Physiol. 108(3): 1185-1195). In animal cells, Enolase has also been known to function as a transcription factor that represses the expression of c-myc by binding to the c-myc gene promoter.
In higher plants, Enolase, like other glycolytic enzymes, is present as multiple isoforms localized to the cytosol and to plastids. In addition to its essential role in glycolysis and gluconeogenesis, in plants, Enolase plays specialized roles in processes with high demand for carbon flux through glycolysis such as fruit ripening (Van Der Straeten et al., 1991; Plant Cell, 3: 719-735) and growth under conditions of anaerobiosis. Exposure of plants to anaerobic stress causes a shift from an oxidative to a fermentative mode of carbohydrate metabolism, resulting in the increased expression of many enzymes of the glycolytic pathway (Lal et al; 1998 Plant Physiol. 1998 December; 118(4):1285-93).
In animal cells, part of the Enolase protein has been shown to bind to the promoter element of the c-myc gene and to repress c-myc expression (Subramanian et al., 2000; J. Biol. Chem., 275: 5958-5965). Similarly a plant derived Enolase protein, the LOS2 protein, can bind to the c-myc promoter as well as to the promoter of the zinc finger STZ/ZAT10 from Arabidopsis. The characteristic DNA binding and repressor protein domains of Enolases are conserved between the human alpha-Enolase and the Arabidopsis LOS2 Enolase. The LOS2 Enolase protein has been suggested to play a role in controlling gene expression under low temperature stress in Arabidopsis thaliana (Lee et al. 2002; EMBO J. 21(11): 2692-2702). The Arabidopsis thaliana los2 mutant plants reportedly displayed chilling and freezing sensitivity.
3. Zn Transporter of Arabidopsis thaliana (ZAT)
Van der Zaal et al., (Plant Physiology, March 1999, Vol. 119, pp. 1047-1055) describe a ZAT zinc transporter (the term ZAT being derived from Zn transporter of Arabidopsis thaliana) of 398 amino acid residues and predicted to have six membrane-spanning domains. The authors analyzed transgenic plants containing the Arabidopsis thaliana ZAT coding sequence under the control of the cauliflower mosaic virus 35S promoter. Plants obtained with ZAT in the sense orientation reportedly exhibited enhanced Zn resistance and strongly increased Zn content in the roots under high Zn exposure. Antisense mRNA-producing plants were reported to be viable, with a wild-type level of Zn resistance and content, like plants expressing a truncated coding sequence lacking the C-terminal domain of the protein.
Ramesh et al., (Plant Molecular Biology 54: 373-385, 2004) describe the effects of overexpression of the Arabidopsis zinc transporter AtZIP1 in Hordeum vulgare cv. Golden Promise on plant growth, seed mineral content and zinc transport rates. The authors reported that in the long-term growth experiments there were no significant differences between transgenic and control lines in leaf zinc content or shoot biomass under zinc-sufficient or zinc-deficient conditions. Root-to-shoot ratios were reported to be higher in the transgenic plants grown under low zinc conditions.
Since the ZIP-type zinc transporters described in Ramesh et al. did not give any significant differences between transgenic and control lines in leaf zinc content or shoot biomass under zinc-sufficient or zinc-deficient conditions, it was surprising to find that ZAT-like zinc transporters gave enhanced yield-related traits upon modulating expression in a plant of a ZAT-like zinc transporter.
4. 6-Phosphogluconate Dehydrogenase (6-PGDH)
6-Phosphogluconate dehydrogenase (EC: 1.1.1.44) (6-PGDH) is an oxidative carboxylase that catalyses the decarboxylating reduction of 6-phosphogluconate into ribulose 5-phosphate in the presence of NADP. This enzyme contributes to generate a significant amount of reducing power (NADPH) in the a cell. This reaction is a component of the hexose mono-phosphate shunt and pentose phosphate pathways (PPP) Broedel and Wolf J. Bacteriol. 172 4023-4031 1990. Prokaryotic and eukaryotic 6PGD are proteins of about 470 amino acids whose sequence are highly conserved Adams et al. EMBO J. 2 1009-1014 1983. The protein is a homodimer in which the monomers act independently: each contains a large, mainly alpha-helical domain and a smaller beta-alpha-beta domain, containing a mixed parallel and anti-parallel 6-stranded beta sheet. NADP is bound in a cleft in the small domain, the substrate binding in an adjacent pocket.